Ractopamine induces differential gene expression in porcine skeletal muscles

A. M. Gunawan, B. T. Richert, A. P. Schinckel, A. L. Grant and D. E. GerrardRactopamine induces differential gene expression in porcine skeletal muscles

published online April 27, 2007J ANIM SCI 

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Running Head: Ractopamine-induced gene expression 123

Ractopamine induces differential gene expression in porcine skeletal muscles 456789

1011121314151617

Gunawan, A. M., B.T. Richert, A.P. Schinckel, A.L. Grant, D.E. Gerrard11819

Department of Animal Sciences, Purdue University, W. Lafayette, IN 47907 20212223242526272829303132333435363738

Purdue University Agricultural Research Programs Journal Paper No. XX,XXX 394041

1Correspondending author: [email protected]). 42

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ABSTRACT: Ractopamine (RAC) improves growth by increasing lean accretion and 43

decreasing fat deposition through repartitioning nutrients from adipose tissue to skeletal 44

muscle. Although not completely understood, RAC alters the proportion of muscle fiber 45

type composition toward a “faster-contracting” phenotype. Because one of the primary 46

determinants of contractile speed is the relative abundance of myosin heavy chain 47

(MyHC) isoform and because the genes encoding these isoforms are transcriptionally 48

regulated, RAC likely alters MyHC gene expression. Using real time PCR, relative 49

transcript abundance of individual type I, IIA, IIX and IIB and total MyHC, as well as 50

glycogen synthase (GS), citrate synthase (CS), lactate dehydrogenase (LDH), peroxisome 51

proliferator activated receptor α (PPARα), β1-adrenergic receptor (AR), and β2-AR were 52

determined in the LM of 44 pigs fed RAC (20 mg/kg) for 0, 1, 2, or 4 wk. In addition, 53

MyHC isoform expression was determined in the LM and red (RST) and white (WST) 54

semitendinosus muscles of 48 pigs fed RAC (20 mg/kg) for shorter periods of 12, 24, 48 55

or 96 h. Type I MyHC expression was unaffected (P > 0.73) by RAC administration. 56

Type IIA MyHC expression decreased (P < 0.0001) by 96 h, was lower (P < 0.0001) by 1 57

wk and returned to normal by 4 wk. Type IIX MyHC mRNA decreased (P < 0.001) by 2 58

wk and continued to decrease (P < 0.0001) by 4 wk. Most interesting was an increase (P 59

< 0.0001) in type IIB MyHC by 12 h which was maintained at an elevated level 60

throughout the 4 wk feeding period. Abundance of GS transcript was increased (P < 61

0.05) similarly by 12 h, but was not different from controls at 2 wk and lower (P < 0.01) 62

at 4 wk. Gene expression of β1-AR was not affected by feeding RAC, whereas β2-AR 63

gene expression was decreased (P < 0.05) by 2 wk. These data show MyHC genes are 64

differentially regulated by RAC and suggest that the beta adrenergic agonists-induced 65

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repartitioning effect is, in part, mediated by changing muscle fiber type-specific gene 66

expression, perhaps through the β2-AR. 67

68

Key words: Fiber type, myosin heavy chain, ractopamine 69

70

INTRODUCTION 71

Initial skeletal muscle gene expression studies suggested beta adrenergic agonists 72

(BAA) universally up-regulated skeletal muscle specific contractile protein. Clearly, 73

various species of myofibrillar mRNA are greater in ractopamine (RAC) -fed pigs (Smith 74

et al., 1989; Helferich et al., 1990; Grant et al., 1993). However, muscle histochemical-75

based data show BAA administration stimulates muscle growth in a fast fiber type-76

specific manner (Moloney et al., 1990; Wheeler and Koohmaraie, 1992), suggesting 77

BAA may differentially alter muscle fiber type-specific MyHC gene expression. 78

Specifically, BAA feeding increases the frequency and size of type II fibers, especially 79

type IIB fibers, the fastest contracting, most glycolytic muscle fiber type (Beermann et 80

al., 1987; Zeman et al., 1988; Polla et al., 2001). In fact, we have shown that the amount 81

of type II IIB MyHC increases at the expense of those isoforms associated with slower-82

twitch, more oxidative fibers suggesting BAA stimulate muscle fibers to transition to a 83

faster phenotype (Depreux et al., 2002). Taken together, these findings are consistent 84

with the idea that improved efficiency in BAA-fed pigs may be a function of more 85

favorable protein turnover rates in whiter muscle (Dadoune et al., 1978). Not only does 86

skeletal muscle become more glycolytic after agonist administration (Eisemann et al., 87

1987; Vestergaard et al., 1994), BAA induce changes in energy-metabolism (Rajab et al., 88

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2000; Polla et al., 2001) suggesting BAA stimulates muscle to change its functional and 89

energetic capabilities. The objective of the present study was to determine the effect of 90

RAC on skeletal muscle fiber type-specific gene expression. 91

92

MATERIALS AND METHODS 93

Overall Design 94

Two separate experiments were conducted to determine the role of RAC on 95

muscle fiber type-specific gene expression. The first study was initially conducted to 96

provide changes in muscle expression on a weekly basis. A subsequent study was 97

conducted to define further the time, within hours, of when these genes were altered, or if 98

there were initial, or transient, changes that occurred within hours of treatment but 99

subsided by 1 wk. 100

Animals. 101

Exp. 1. Ractopamine-HCl (Elanco Animal Health, IN) was administered at 20 102

ppm daily in feed for either 0, 1, 2 or 4 wk to 44 pigs (average BW 90 ± 10 kg) obtained 103

from Purdue University Animal Sciences Research and Education Center. Diets were 104

corn and soybean meal based and formulated to meet or exceed all nutrients requirements 105

of these pigs (NRC, 1998). Diets were formulated to 1.15% Lys, 19.5% CP and 5.0% 106

supplemental fat, with or without RAC. Control pigs (barrows and gilts) were 107

slaughtered after a 4 wk period of growth, whereas pigs that were fed RAC for 1 or 2 wk 108

followed a 3 or 2 wk period of growth, respectively. This was an attempt to achieve the 109

predetermined duration of feeding (4 wk for all pigs), and target an average slaughter 110

weight of 115 kg. Pigs were processed according to normal industry procedures and LM 111

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samples were taken immediately post-exsanguination. Samples were frozen in liquid 112

nitrogen and stored at -80°C. The Purdue Animal Care and Use Committee approved all 113

procedures for the care and use of pigs. 114

Exp. 2. In a second study, forty-eight pigs (barrows and gilts; average BW 98 ± 115

3 kg) obtained from Purdue University Animal Sciences Research and Education Center 116

and used to titrate the time needed to up-regulate muscle fiber type-specific gene 117

expression. When pigs reached an average BW of 90 kg, pigs were blocked by initial 118

BW into 2 groups. Within these two groups, equal numbers of pens were randomly 119

assigned to treatments of 0 or 20 ppm RAC (Elanco Animal Health) for a total of 16 120

pens. Both diets were corn and soybean meal based and formulated to meet or exceed all 121

nutrients requirements of these pigs (NRC, 1998). Diets were formulated to 1.15% Lys, 122

19.5% CP and 5.0% supplemental fat, with or without RAC. Following 12, 24, 48 or 96 123

h, 6 pigs were harvested for each level of RAC, for a total of 48 pigs. Pigs were 124

processed according to normal industry procedures and red (RST) and white 125

semitendinosus (WST), and LM samples were taken immediately post-exsanguination. 126

Samples were frozen in liquid nitrogen and stored at -80°C. The Purdue Animal Care 127

and Use Committee approved all procedures for the care and use of pigs. 128

129

Total RNA Preparation. 130

Total RNA was extracted from porcine skeletal muscle using the single-step 131

method (Chomczynski and Sacchi, 1987) with modifications. Briefly, 100 mg of skeletal 132

muscle powdered in liquid nitrogen was placed into 3 mL of Solution D, composed of 4 133

M guanidium isothiocyanate (Sigma, St. Louis, MO), 25 mM sodium citrate (pH 7), 0.5 134

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% sarcosyl, and 0.1 M 2-mercaptoethanol, in a 50-mL conical tube held on ice. Tissue 135

was homogenized using a Polytron Homogenizer (Brinkman Instruments, New York, 136

NY) at speed 6, then transferred to a fresh 15-mL conical tube. Next, 300 µL of 2 M 137

sodium acetate (pH 4) was added and vortexed. Protein and lipids in the preparation were 138

extracted by sequentially adding 3 mL phenol saturated with diethyl pyrocarbonate-139

treated water (pH 4.3), and 600 µL chloroform-isoamylalcohol (40:1 vol/vol). After 140

thorough vortexing, tubes were placed on ice for 15 min. Samples were centrifuged at 141

10,000 x g at 4°C for 20 min to separate aqueous and organic phases. The aqueous phase 142

(upper) was carefully transferred to a fresh 15-mL conical tube. Attention was made to 143

avoid disturbing the interphase. Precipitation of the RNA was facilitated by adding 3 mL 144

isopropanol and holding samples at -20°C for 1 h. Precipitated RNA was then collected 145

by centrifugation at 10,000 x g at 4°C for 30 min. Pellets were then redissolved in 0.9 146

mL solution D, 0.9 mL isopropanol was added and RNA was allowed to precipitate at -147

20°C for 1 h. Precipitated RNA was then collected by centrifugation at 21,000 x g at 4°C 148

for 30 min. Isopropanol was discarded, and the pellet was washed twice with 1 mL 75% 149

ethanol (vol/vol). Pellets were air dried for 10 min and resuspended in 50 µL of TE-8, 150

composed of 1 M tris-HCl (pH 8), 0.2 M EDTA (pH 8), and 0.1 M diethyl 151

pyrocarbonate-treated water. 152

153

RNA Quantification. 154

Total RNA isolated from skeletal muscle was measured using the Ribogreen 155

quantification kit (Molecular Probes, Eugene, OR). RNA was first treated with 4 U 156

recombinant DNAse (Ambion, Indianapolis, IN) in 2 µL of a 10 X DNase buffer 157

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containing 100 mM Tris-HCl, pH 7.5; 25 mM MgCl2; 5 mM CaCl2; and 3 µL nuclease-158

free water (Ambion) and digested for 30 min at 37°C. DNase Inactivation Reagent (5 µL; 159

Ambion), a resin that binds DNase, was added to the reaction and mixed by flicking the 160

tubes. RNA was then loaded into 0.22 µm Spin-X Centrifuge Tube Filters (VWR 161

International, West Chester, PA) and centrifuged at 10,000 x g for 1 min. A 1-µL sample 162

of purified RNA was diluted with 249 µL TE-buffer containing 200 mM Tris-HCl, 20 163

mM EDTA (pH 7.5; Ambion). Next, 75 µL TE-buffer and 100 µL Ribogreen reagent 164

were combined in each well on a 96-well microplate. Prepared RNA samples 25 µL) 165

were added to the reagents on the microplate. A GENios Pro fluorometer (Tecan, 166

Durham, NC) was used to measure the fluorescent signal at 480 (excitation) and 520 nm 167

(emission). 168

169

cDNA Synthesis 170

RNA was reverse transcribed to cDNA. A cDNA master mix was prepared from 171

5 U Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen, 172

Carlsbad, CA), 0.5 U SUPERase-In (Ambion), 10 mM DTT (Invitrogen), and 5X First 173

strand buffer (Invitrogen). Total RNA (0.5 µg) in 5 µL nuclease-free water was added to 174

100 ng/µL random hexamers and 100 µM each dNTP. Samples were denatured at 65 °C 175

for 5 min and then chilled to 4 °C on ice. Four µL cDNA master mix was added to 176

denatured RNA and sequentially incubated at 25 °C for 10 min, 37 °C for 50 min, 70 °C 177

for 10 min, and chilled to 4 °C on ice. Finally, cDNA samples were diluted with 178

nuclease-free water and aliquoted so that gene quantification was based on 50 ng of total 179

RNA per 5 µL. 180

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181

Real Time PCR 182

Exp. 1. Relative transcript abundance was determined using real time polymerase 183

chain reaction (qPCR). Primer sequences for individual myosin heavy chain (MyHC) 184

isoforms I, IIA, IIX, and IIB, total MyHC, GS, CS, PPARα, β-ARs 1 and 2 and β-actin, 185

are shown in Table 1. Individual MyHC primers targeted the 5’-untranslated region of 186

their respective cDNA (da Costa et al., 2002), convey isoform specificity. Total MyHC 187

primers targeted the S1 loop region, where homology among MyHC isoforms is 188

essentially identical. Real time PCR was carried out according to optimized gene specific 189

protocols, shown in Table 2. The first cycle in the log linear region of amplification 190

where a significant increase in fluorescence was detected above background was 191

designated the threshold cycle (Ct). All expression data were normalized to β-actin. 192

Amplification of highly expressed genes, therefore were detected at a lower cycle number 193

than those genes expressed at relatively low level, which require additional cycles; 194

therefore, Ct and gene expression are inversely related. 195

Exp. 2. Primer sequences for adult MyHC isoforms, GS, CS, β-AR 1 and 2, and 196

β-actin are described above. Quantification standards were composed of aliquots of PCR 197

products in 10-fold serial dilutions ranging from 109 to 101 molecules. Standards were 198

amplified in triplicate, and used to calculate a regression of threshold cycle on molecule 199

copy number to determine a log value of starting abundance for each cDNA aliquot, 200

amplified in duplicate, based on individual threshold cycle. PCR reactions were run for 201

40 cycles in an iCycler Real-Time PCR Detection System (Bio-Rad Inc., Hercules, CA) 202

according to optimized gene specific protocols. Amplification to the fluorescence 203

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threshold occurs sooner for highly expressed genes than those genes expressed at 204

relatively low levels. In the case of this experiment therefore, a large log value indicates 205

high gene expression, and a small log value indicates low expression of a gene. 206

207

Statistical Analysis 208

Analysis of variance was generated by using the GLM procedure of SAS (SAS 209

Inst., Inc., Cary, NC) with week and RAC as the main effects. Least square means and 210

average SEM were calculated for all Ct (Exp. 1) or log starting abundance (Exp. 2) data 211

and the Student-Newman-Keuls procedure of SAS was used to separate means when a212

significant F-test (P < 0.05) was observed. 213

214

RESULTS 215

Exp. 1. Real time PCR was used to determine the effect of RAC on gene 216

expression in porcine skeletal muscle following a period of 0, 1, 2 or 4 wk of feeding 217

RAC. Beta-actin, did not differ (P > 0.05) over the course of the experiment (data not 218

shown). Expression of the type I MyHC gene was not altered by RAC during the entire 219

length of the study (Figure 1A). Type IIA MyHC gene expression decreased (P < 220

0.0001) by 1 wk of RAC administration, remained lower (P < 0.001) at 2 wk, but was not 221

different from controls by 4 wk (Figure 1B). Ractopamine did not influence MyHC type 222

IIX gene expression by 1 wk, but decreased (P < 0.001) by 2 and 4 wk (Figure 1C). 223

Expression of MyHC type IIB was increased (P < 0.0001) by 1 wk, and remained 224

elevated throughout the remainder of the study (Figure 1D). Ractopamine increased (P < 225

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0.001) total MyHC transcript abundance following 1 and 2 wk administration, yet 226

expression did not differ from controls at 4 wk (Figure 1E). 227

Citrate synthase (CS) gene expression was not different from controls at 1 wk, but 228

decreased (P < 0.01) by 2 and 4 wk (Figure 2A). Glycogen synthase (GS) mRNA 229

abundance was greater (P < 0.05) by 1 wk of RAC administration, returned to the level of 230

control at wk 2, and decreased (P < 0.01) by 4 wk (Figure 2B). Lactate dehydrogenase 231

(LDH) gene expression was not affected by RAC (Figure 2C). Expression of the PPARα232

gene decreased (P < 0.05) by 1 wk of RAC administration, but was not different from 233

controls by 2 and 4 wk (Figure 2D). Ractopamine had no effect on β1-AR gene 234

expression (Figure 2E), however, β2-AR gene expression decreased (P < 0.05) by 2 and 4 235

wk (Figure 2F). 236

Exp. 2. β-Actin gene expression, a control to test for experimental variation, was 237

not affected by RAC (data not shown). Compared with LM muscles, MyHC type I gene 238

expression was greater (P < 0.0001) in RST and lower (P < 0.0001) in WST (data not 239

shown). Type IIA MyHC gene expression was also greatest (P < 0.0001) in RST 240

muscles, but least (P < 0.0001) in LM muscles (data not shown). Ractopamine decreased 241

(P < 0.0001) type IIA MyHC gene expression by 96 h compared with controls (Figure 3). 242

No muscle X time interaction was observed. Ractopamine did not affect type IIX MyHC 243

prior to 96 h, but gene expression was greater (P < 0.01) in WST than RST muscles, 244

whereas LM muscles had least (P < 0.01) type IIX MyHC expression (data not shown). 245

RST muscles had lower (P < 0.0001) type IIB MyHC gene expression than WST and LM 246

muscles (data not shown). However, RAC increased (P < 0.0001) type IIB MyHC gene 247

expression in all muscles by 12 h and maintained this level of expression at all 248

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subsequent time points in the study (Fig 4A). Glycogen synthase gene expression was 249

greater (P < 0.05) in WST and LM muscles than RST muscles (data not shown). 250

Furthermore, expression was increased (P < 0.05) by 12 h after RAC administration in all 251

muscles studied (Figure 4B). Gene expression of CS was not different among muscles 252

and was unaffected by RAC in our study (data not shown). Gene expression of β1-AR 253

did not vary with treatment or muscle prior to 96 h (data not shown) whereas RST 254

muscles had greater (P < 0.01) β2-AR gene expression than WST and LM but likewise, 255

did not change within 96 h of treatment (data not shown). 256

257

DISCUSSION 258

Several groups have reported detecting increased total RNA and protein in muscle 259

of BAA treated animals (Helferich et al., 1990; Koohmaraie et al., 1991; Grant et al., 260

1993), however, little data exist regarding the expression of multiple genes within a 261

larger gene family, like myosin and its associated isoforms. Data from these studies 262

show that RAC feeding differentially alters fiber type-specific gene expression rather 263

than globally increasing the expression of all contractile proteins. 264

In our studies, type I MyHC gene expression was not affected by RAC feeding. 265

These results are supported by earlier data indicating muscle hypertrophy and subsequent 266

increases in muscle mass of those animals fed BAA generally lack increases in type I 267

MyHC. Using histochemical approaches, Beermann et al. (1987) showed that type I 268

fibers contributed little to cimaterol-induced hypertrophy in the biceps femoris, 269

semitendinosus, and semimembranosus muscles of lambs. This was further verified in a 270

separate lamb study by Kim et al. (1987a). Furthermore, Aalhus et al. (1992) reported 271

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RAC did not significantly alter the cross sectional area of type I muscle fibers in pig 272

muscle, while others have reported a reduction in these fibers in the biceps femoris of 273

pigs fed salbutamol (Oksbjerg et al., 1994). Using a whole muscle enzyme linked 274

ELISA-based assay, we showed that even feeding as much as 60 ppm RAC for up to 42 d 275

does not change the relative abundance of type I MyHC protein in the red and white 276

semitendinosus muscle or LM of pigs (Depreux et al., 2002). The transient (2 wk) 277

increase in total MyHC message observed in porcine muscle after BAA feeding suggests 278

the proportion of type I MyHC may have increased slightly for 2 wk; however, this 279

would be difficult to resolve given the aforementioned literature. 280

Type IIA MyHC gene expression first decreased between 48 and 96 h after RAC 281

administration and continued to decrease to its lowest level by 1 wk, but returned to 282

control levels by 4 wk. This pattern of gene expression represents a rapid down-283

regulation of the type IIA gene followed by a gradual recovery to full, pre-treatment 284

levels. During the same time, however, type IIX MyHC gene expression decreased 285

throughout the entire duration of the study while increased type IIB MyHC gene 286

expression was initially observed to increase by 12 h and remained elevated through the 4 287

wk feeding period. The majority of existing muscle fiber type data suggests that BAA-288

induced muscle hypertrophy results from increases in cross sectional area of type II fibers 289

(Beermann et al., 1987; Zeman et al., 1988). Consistent with our results, several 290

researchers have shown that the percentage of type IIA fibers decreases with BAA 291

feeding while type IIB increases (Aalhus et al., 1992; Vestergaard et al., 1994), 292

suggesting the increased frequency of type IIB fibers in skeletal muscle may be 293

responsible for BAA-improved growth (Wu et al., 1986; Beermann et al., 1987), albeit no 294

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cause and effect relationship has ever been established between the increase in muscle 295

growth and the frequency, or size, of type IIB. Unfortunately, early studies were plagued 296

by limitations in the specificity of fiber typing protocols (Lefaucheur et al., 2004). 297

Muscle fibers histochemically identified as type IIB are composed of both type IIX or IIB 298

MyHC and therefore, changes in fiber type composition may have been obscured by 299

inclusion of different fiber types responding differently to treatments (Lefaucheur et al., 300

1998). In addition, means of reporting fiber type data vary from those reporting absolute 301

frequencies to those reporting fiber type on a per cross-sectional area basis. Although 302

both approaches are valid, it is difficult to collectively interpret these data. Data reported 303

herein reflect whole muscle gene expression and essentially reflect data reported by 304

Depreux et al. (2002) that RAC increases relative abundance of type IIB MyHC at the 305

expense of type IIA and IIX using whole muscle MyHC preparations (Depreux et al., 306

2002). Even though variability in the antibodies used to quantify type IIA and IIX 307

MyHC in that study have been identified (Lefaucheur et al., 2004), the fact that 308

immunoreactivity to MyHCs decreased while increases in IIB antigenicity was observed 309

argues muscle becomes “faster” with BAA feeding at the expense of more oxidative, fast 310

contracting fibers, namely IIA and IIX. 311

The enhanced protein synthesis observed in muscle cells cultured in the presence 312

of BAA is blocked by compounds that bind specifically to the beta-adrenergic receptors 313

(β-AR) (Anderson et al., 1990). To date, 3 β-AR (β1, β2 and β3) are known to exist in 314

porcine skeletal muscle and represent 60, 39 and 0.7 percent, respectively, of the total β-315

AR on muscle cells (McNeel and Mersmann, 1999). We were unable to detect any β3-316

AR signal in the skeletal muscles studied using real time PCR. The fact that we did not 317

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detect this species of β-AR does not imply that it does not play an important role in 318

modulating BAA effect in pigs. However, given our primers were specifically designed 319

to porcine sequences, this simply indicates it is expressed at extremely low levels in 320

skeletal muscle. The β1-AR, however, was easily detected in porcine skeletal muscle but 321

significant changes were not noted. On the other hand, β2-AR gene expression decreased 322

significantly by 2 wk. Interpretation of these data is somewhat difficult given receptor 323

binding studies were not conducted. However, if reflective of receptor numbers and 324

binding, this suggests a portion of the loss of response to BAA feeding (Williams et al., 325

1994; Schinckel et al., 2003a) may be mediated through loss of β2-AR. RAC was 326

originally thought to be selective for the β1-AR subtype (Smith et al., 1990; Moody et al., 327

2000). However, Mills (2002) suggested that BAA-induced activation of the cAMP 328

signaling cascade is more efficient through the β2-AR even though RAC binds β1- and β2-329

AR with similar affinities (Spurlock et al., 1993; Liang et al., 2000; Mills, 2002). Kim et 330

al. (1992) postulated the same after observing that β-AR binding was reduced in the 331

skeletal muscle of cimaterol fed mice prior to the loss of growth-stimulation. Spurlock et 332

al. (1994), however, showed no loss in receptor number in muscle of pigs fed RAC for up 333

to 4 wks. These discrepancies may be related to a combination of post-transcriptional 334

events and species differences. Curiously, the β-AR are predominantly found in slow-335

twitch, oxidative muscles (Williams et al., 1984). Muscle fibers are extremely sensitive 336

to hormonal or functional cues. In particular, muscle fiber type transitions occur along a 337

well-characterized pathway (I↔IIA↔IIX↔IIB; Pette and Staron, 1997). Even though 338

our data are not based on individual fiber data and, therefore, cannot specifically address 339

this pathway, per se, classical histochemical data firmly support the idea that BAA 340

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stimulation forces muscle to a faster-contracting phenotype. Therefore, it is possible that 341

the loss of the β2-AR may simply reflect the loss of slow fibers, which have greater β-AR 342

expression. However, our data does not support this mechanism because we failed to 343

observe a muscle X time interaction for β2-AR expression in Exp. 2, which would be 344

expected given that greater expression is observed in the red portions of the 345

semitendinosus. In addition, it is difficult to envision that changes in type IIA fibers 346

would be enough to change whole muscle β2-AR expression given IIA fibers represent 347

only a small fraction (< 10%; Lefaucheur et al., 2004) of the total fibers in the LM. 348

Further studies will be required to understand fully how various β-AR mediate this 349

response, and more importantly, which fiber type (within the type II fibers), if any, 350

respond directly to BAA feeding. 351

Muscles possessing different amounts and types of MyHC inherently contain 352

different types of energy metabolisms to facilitate their function (Gauthier, 1969). A 353

slow-to-fast transition of MyHC isoforms induced by clenbuterol is associated with 354

decreases in the activity of oxidative enzymes and increases in activity of glycolytic 355

enzymes in fast- and slow-twitch muscles (Polla et al., 2001). It is important to note, 356

however, that enzyme activities are generally controlled by substrate availability or other 357

types of posttranscriptional modifications and therefore, need not correlate with CS 358

synthase mRNA levels. We did evaluate enzyme activity in our studies. Nonetheless, 359

CS gene expression was decreased by RAC administration in our study by 2 and 4 wk. 360

Our data are consistent with a BAA-induced decrease in aerobic enzyme activity in cattle 361

and pigs indicating a shift toward the more glycolytic type metabolism that accompanies 362

the transitioning fiber type profile in muscle of BAA treated animals (Zimmerli and 363

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Blum, 1990; Vestergaard et al., 1994). Curiously, GS gene expression increased by 12 h 364

of RAC administration, returned to the level of controls by 2 wk, and further decreased 365

by 4 wk where expression was significantly less than controls. Glycogen synthase is the 366

rate-limiting enzyme for synthesis of glycogen, a major energy storage molecule in the 367

liver and skeletal muscle. Highly-selected pigs with a high rate of lean gain have 368

intrinsically greater glycolytic metabolism which can be enhanced by BAA 369

administration (Oksbjerg et al., 1990). Of course, glycogenolysis is stimulated by acute 370

administration of epinephrine [5 to 20 min; (Richter et al., 1982)]. Epinephrine enhances 371

glycogenolysis in skeletal muscle and glucose production from the liver as a result of 372

direct stimulation of glycogenolysis and indirect stimulation of gluconeogenesis, 373

respectively (Arinze and Kawai, 1983). Therefore, mobilization of energy stores from 374

liver into the circulation by acute administration of BAA increases glucose availability to 375

skeletal muscle (Mersmann et al., 1987; Assimacopoulos-Jeannet et al., 1991) and this 376

cytosolic increase of glucose probably stimulates GS expression. Our data are consistent 377

with these reports suggesting GS gene expression is increased by BAA acutely in an 378

attempt to accommodate the increased glucose rapidly entering the cell. Afterwards, 379

however, it is possible that chronic administration of RAC leads to depleted levels of 380

glucose, most likely due to increased energy needs associated with increased muscle 381

growth. As a result, less GS would be required, leading to down-regulation of the gene. 382

Although somewhat less reflective of fiber differences, these data provide insight into 383

how BAA may enhance muscle growth. 384

Surprisingly, LDH gene expression was not significantly increased in our studies 385

as BAA administration stimulates lactate production in sheep and pigs (Warriss et al., 386

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1989; Warriss et al., 1990). Clearly, LDH activity, an indicator of glycolytic energy 387

metabolism, is increased by BAA feeding and corresponds with increased CSA of fast-388

twitch fibers (Pellegrino et al., 2004). Jungman et al. (1983) showed that β-AR-mediated 389

signal transduction pathways transcriptionally regulate LDH expression (Jungmann et al., 390

1983). However, expression of LDH may be at an extremely high level in LM, 391

demonstrating a further increase in gene expression may not be possible to detect. 392

Ractopamine decreased PPARα gene expression by 1 wk. Furthermore, PPARα,393

a ligand-activated nuclear hormone receptor first identified in mice (Issemann and Green, 394

1990), regulates lipid metabolism by stimulating expression of genes encoding enzymes 395

required for peroxisomal β-oxidation (Kliewer et al., 1994). The PPARα activates 396

transcription by binding to peroxisome proliferator response elements in the DNA 397

sequence, and is abundantly expressed in tissues with high rates of β-oxidation including 398

liver, kidney, heart, and skeletal muscle, promoting cellular uptake and oxidation of fatty 399

acids (Kliewer et al., 1994; Braissant et al., 1996; Mukherjee et al., 2000). As mentioned 400

above, RAC immediately mobilizes glucose, which likely reduces fatty acid oxidation 401

and those associated enzymes. Of course, and consistent with the decreased need for GS, 402

PPARα returns to normal by 2 wk and is maintained at this level at 4 wk, suggesting 403

changes in β-oxidation may be only indirectly associated with BAA feeding but clearly is 404

consistent with reduced adipose tissue mass in BAA fed animals (Crenshaw et al., 1987). 405

Data presented herein suggest RAC differentially induces expression of the type 406

IIB MyHC gene at the expense of the other isforms. However, RAC was administered to 407

pigs during the finishing phase where pigs normally exhibit muscle hypertrophy. Thus, it 408

cannot be over stated that it remains highly possible that muscle assumes the 409

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aforementioned characteristics simply as a result of it growing faster or experiencing 410

hypertrophy. Type II fibers are larger, more glycolytic and exist in higher frequencies in 411

muscles that experience the greatest amount of hypertrophy in a growing animal. 412

Moreover, lines of pigs highly-selected for augmented muscle growth contain more type 413

II fibers and express greater amounts of type IIB MyHC (Oksbjerg et al., 1990; Karlsson 414

et al., 1993). Therefore, muscle hypertrophy may stimulate transitions to the faster 415

contracting, more glycolytic type II fiber phenotype, rather than vice versa. Additional 416

studies will be necessary to document whether there is a true cause and effect relationship 417

between muscle fiber type and muscle growth or simply reflects a close association 418

between the whole animal and its underlying physiology. 419

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LITERATURE CITED 420 421

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426 Anderson, P. T., W. G. Helferich, L. C. Parkhill, R. A. Merkel, and W. G. Bergen. 1990. 427

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430 Arinze, I., and Y. Kawai. 1983. Adrenergic regulation of glycogenolysis in isolated 431

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442 Braissant, O., F. Foufelle, C. Scotto, M. Dauca, and W. Wahli. 1996. Differential 443

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474 Helferich, W. G., D. B. Jump, D. B. Anderson, D. M. Skjaerlund, R. A. Merkel, and W. 475

G. Bergen. 1990. Skeletal muscle alpha-actin synthesis is increased 476pretranslationally in pigs fed the phenethanolamine ractopamine. Endocrinology 477126:3096-3100. 478

479 Jungmann, R., D. Kelley, M. Miles, and D. Milkowski. 1983. Cyclic AMP regulation of 480

lactate dehydrogenase. Isoproterenol and N6,O2- dibutyryl cyclic amp increase 481the rate of transcription and change the stability of lactate dehydrogenase a 482subunit messenger RNA in rat C6 glioma cells. J. Biol. Chem. 258:5312-5318. 483

484 Kim, Y. S., Y. B. Lee, and C. R. Ashmore. 1988. Cimaterol-induced growth in rats: 485

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496 Koohmaraie, M., S. D. Shackelford, N. E. Muggli-Cockett, and R. T. Stone. 1991. Effect 497

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504 Liang, W., C. A. Bidwell, P. R. Collodi, and S. E. Mills. 2000. Expression of the porcine 505

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Zimmerli, U. V., and J. W. Blum. 1990. Acute and longterm metabolic, endocrine, 598respiratory, cardiac and skeletal muscle activity changes in response to perorally 599administered β-adrenoceptor agonists in calves. J. Anim. Physiol. Anim. Nutr. 60063:157-172. 601

602

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Table 1. Primer sequences 603

Gene Primer Sequence (5'-3') PCR product (bp)

Type I MyHC1

Forward (MyHC I-F) GGC CCC TTC CAG CTT GA 63

Reverse (MyHC I-R) TGG CTG CGC CTT GGT TT

Type IIA MyHC1

Forward (MyHC IIA-F) TTA AAA AGC TCC AAG AAC TGT TTC A 109

Reverse (MyHC IIA-R) CCA TTT CCT GGT CGG AAC TC

Type IIX MyHC1

Forward (MyHC IIX-F) AGC TTC AAG TTC TGC CCC ACT 79

Reverse (MyHC IIX-R) GGC TGC GGG TTA TTG ATG G

Type IIB MyHC1

Forward (MyHC IIB-F) CAC TTT AAG TAG TTG TCT GCC TTG AG 83

Reverse (MyHC IIB-R) GGC AGC AGG GCA CTA GAT GT

Total Myosin Heavy Chain

Forward (MyHC-F) TTC AAC CAC CAC ATG TTC GTG 126

Reverse (MyHC-R) GAT GCC CAT GGG CTT CTC GAT

Glycogen Synthase

Forward (Gly-F) CCC AGT GGG AGG AGG CAG TCT TG 122

Reverse (Gly-R) GAA CCG CCG GTC CAG AAT GTA GA

Citrate Synthase

Forward (Cit-F) TGC CAG TGC TTC TTC CAC GAA CTT 97

Reverse (Cit-R) GTT GCC GTG TTG CTG CCT GAA G

Lactate Dehydrogenase

Forward (LDH-F) GTG CAT CCG ATT TCC ACC ATG ATT 89

Reverse (LDH-R) CCA TTC TGT CCC AAG ATG CAA GGA

604

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Table 1 (Continued). Primer sequences 605606

Gene Primer Sequence (5'-3') PCR product (bp)

Peroxisome Proliferator Acitvated Receptor α

Forward (PPARα-F) CAA GGG CTT CTT TCG GAG AAC CAT 160

Reverse (PPARα-R) GCG CCC AAA TCG AAT TGC ATT AT

β-Adrenergic Receptor 1

Forward (β1AR-F) CTG CGA AGA CTA GGG AAG GGA TGG 83

Reverse (β1AR-R) CCC CGG GAA CGG AAT GGA A

β-Adrenergic Receptor 2

Forward (β2AR-F) GGC TGC CCT TCT TCA TCG TCA AC 90

Reverse (β2AR-R) AGC CCA CCC AGT TTA GCA GGA TGT

β-Actin

Forward (β-Act-F) GGC ATC CAC GAG ACC ACC TTC AA 85

Reverse (β-Act-R) CAG ACA GCA CCG TGT TGG CGT AGA

607 608

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Table 2. Real time polymerase chain reaction conditions 609610

Gene Hot Start Melting Annealing Synthesis Cycles

MyHC Type I 95°C 15 sec 79°C 15 sec 72°C 30 sec 55°C 15 sec 40

MyHC Type IIA 95°C 15 sec 78°C 15 sec 72°C 30 sec 52°C 15 sec 40

MyHC Type IIX 95°C 15 sec 79°C 15 sec 72°C 30 sec 56°C 15 sec 40

MyHC Type IIB 95°C 15 sec 78°C 15 sec 72°C 30 sec 56°C 15 sec 40

Total Myosin Heavy Chain 95°C 15 sec 82°C 15 sec 72°C 30 sec 53°C 15 sec 40

Glycogen Synthase 95°C 15 sec 83°C 15 sec 72°C 30 sec 59°C 15 sec 40

Citrate Synthase 95°C 15 sec 78°C 15 sec 72°C 30 sec 59°C 15 sec 40

Lactate Dehydrogenase 95°C 15 sec 76°C 15 sec 72°C 30 sec 56°C 15 sec 40

Peroxisome Proliferator

Activated Receptor α 95°C 15 sec 80°C 15 sec 72°C 30 sec 54°C 15 sec 40

β-Adrenergic Receptor 1 95°C 15 sec 78°C 15 sec 72°C 30 sec 58°C 15 sec 40

β-Adrenergic Receptor 2 95°C 15 sec 80°C 15 sec 72°C 30 sec 58°C 15 sec 40

β-Actin 95°C 15 sec 80°C 15 sec 72°C 30 sec 59°C 15 sec 40

611612613614615616617618619620621622623624625626627628

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Figure legends 629

Figure 1. Effect of time (wk) of ractopamine (20 ppm) administration on relative type I (A), IIA 630

(B), IIX (C), IIB (D) and total myosin heavy chain (MyHC; E) gene expression normalized to β-631

actin (E; Ct, threshold cycle) in porcine LM (0 and 2 wk, n = 10; 1 and 4 wk, n = 12). Means 632

bearing different letters differ (P < 0.05). 633

634

Figure 2. Effect of time (wk) of ractopamine (20 ppm) administration on relative citrate synthase 635

(A), glycogen synthase (B), lactate dehydrogenase gene expression (C), peroxisome proliferator 636

activated receptor α (D), β1-adrenergic receptor (E) and β2-adrenergic receptor gene expression 637

normalized to β-actin (F; Ct, threshold cycle) in porcine LM (0 and 2 wk, n = 10; 1 and 4 wk, n = 638

12). Means bearing different letters differ (P < 0.05). 639

Figure 3. Effect of time (h) of ractopamine (20 ppm) administration on type IIA myosin heavy 640

chain (MyHC) gene expression (log starting abundance) in the red and white semitendinosus 641

muscle and LM. Each bar is the mean value (+ SE) of 3 muscles from 6 pigs. Means bearing 642

different letters differ (P < 0.0001). 643

644

Figure 4. Effect of ractopamine (20 ppm) administration on: A) type IIB myosin heavy chain 645

(MyHC) and B) glycogen synthase gene expressions (log starting abundance) in the red and 646

white semitendinosus and longissimus muscle. Each bar is the mean value (+ SE) of 3 muscles 647

from 24 pigs (n = 72). Means bearing different letters differ (P < 0.0001). 648

649

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A)

19

20

21

22

23

24

a aa

a

B)

19

20

21

22

23

24

a

a

b

b

C)

15

16

17 c

bc

a

a

D)

12

13

14

15

16

a

b

a a

E)

13

13.5

14

14.5

15

0 1 2 4

Weeks

ab

a

a

ab

Figure 1. 650

Cyc

leTh

resh

old

(Ct)

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A)

18

19

20

21

22

a

b b

a

B)

26

27

28

29

30c

a

bb

C)

14

15

16

17

18

19

a

aa a

D)

25

26

27

28

29

a

ab

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E)

32

33

34

35

36

37

aa

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F)

25

26

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28

0 1 2 4

Ractopamine (Weeks)

b

a

ab

b

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old

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651Figure 2. 652

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653

4

4.5

5

5.5

0 ppm 20 ppm 0 ppm 20 ppm 0 ppm 20 ppm 0 ppm 20 ppm

Log

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12 h 24 h 48 h 96 h

a

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Figure 3. 677678679680681682683

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684685

A)

4

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5.5

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6.5

7

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b

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708709710711712713714715716717718719720721722723

Figure 4. 724

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Citations

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